Islet Amyloid Polypeptide (Amylin) Is Expressed in Sensory Neurons

The Journal of Neuroscience, November 1995, 75(11): 7625-7632

Hindrik Mulder,’ Arnold Leckstriim,* Rolf Uddman,3 Eva Ekblad,’ Per Westermark: and Frank SundIet-’ ‘Department of Medical Cell Research, University of Lund, *Department of Pathology, University Hospital, LinkQping, and 3Department of Otolaryngology at Malm6 General Hospital, University of Lund, Sweden

Islet amyloid polypeptide (IAPP) or amylin is a hormone predominantly expressed in insulin cells (Johnson et al., 1988), candidate predominantly expressed in insulin cells. A role where it is stored in the same secretory granules as insulin (Lu- for IAPP in the regulation of glucose homeostasis and the kinius et al., 1989). The physiological role of IAPP has not yet development of non-insulin-dependent diabetes mellitus been resolved. However, the data available suggest a role for has been proposed. IAPP is structurally related to the sen- IAPP in glucose homeostasis: IAPP constrains insulin-mediated sory neuropeptide calcitonin gene-related peptide. In the glycogenesis in skeletal muscle (Leighton and Cooper, 1988; present study, using in situ hybridization, immunocyto- Frontoni et al., 1991) and inhibits insulin secretion (Wang et al., chemistry, and immunochemistry, the expression of IAPP 1993; Fiirnsinn et al., 1994). in sensory neurons in the rat was investigated. IAPP was IAPP bears an approximately 50% structural homology with expressed in a population of small- to medium-sized nerve calcitonin gene-related peptide (CGRP) (Westermark et al., cell bodies in dorsal root ganglia from all levels and in the 1986; Cooper et al., 1987), which is widely expressed in the jugular-nodose and trigeminal ganglion; IAPP-expressing CNS and PNS, the sensory nervous system in particular (Sundler nerve cell bodies constituted a subpopulation of those ex- et al., 1985; Ishida-Yamamoto and Tohyama, 1989). The genes pressing calcitonin gene-related peptide. In addition, IAPP- for IAPP and CGRP are located on chromosomes 12 and 11, like immunoreactivity occurred in nerve cell bodies storing respectively, and are probably the result of a duplication of an substance P and pituitary adenylate cyclase-activating ancestral gene (Hoppener et al., 1984; Nishi et al., 1989). polypeptide. IAPP-immunoreactive nerve fibers were en- Against this background, it is tempting to speculate that IAPP countered in the dorsal horns of the spinal cord, and to a is also expressed in the nervous system. In fact, IAPP mRNA lesser extent in peripheral tissues receiving sensory inner- has been demonstrated in extracts from rat dorsal root ganglia vation; IAPP-immunoreactive fibers constituted a subpop- (Ferrier et al., 1989; Nicholl et al., 1992) and, recently, it was ulation of those containing calcitonin gene-related peptide shown that in the chicken IAPP is predominantly expressed in and/or substance P. The immunochemical determinations the brain (Fan et al., 1994). However, the cellular expression demonstrated a low level of IAPP-like immunoreactivity in and distribution of IAPP in the sensory nervous system are not the dorsal root ganglia and spinal cord, which chromato- known. Therefore, by use of in situ hybridization, immunocy- graphically coeluted with authentic rat IAPP. We conclude tochemistry, and immunochemistry, we have investigated the that IAPP is expressed in sensory neurons, thus being a expression of IAPP in sensory ganglia and its occurrence in the novel sensory neuropeptide. candidate for which a physi- spinal cord and neuronal elements of peripheral tissues in the ological role remains to be identified. rat. [Key words: islet amyloid polypeptide, amylin, calcitonin gene-related peptide, neuropeptides, sensory nervous sys- Materials and Methods tem, in situ hybridization, immunocytochemistry, radioim- Tissue. Adult female Sprague-Dawley rats (n = 10) were used; they munoassay (R/A)] were anesthetized by pentobarbitone and killed by exsanguination. Dor- sal root ganglia from the levels C, to L,, jugular-nodose, and trigeminal Islet amyloid polypeptide (IAPP), also designated amylin, is the ganglia were rapidly dissected. The following tissues were investigated main constituent of amyloid formed in insulinomas or in islets for the possible occurrence of IAPP-containing nerve fibers: nose skin, tongue, foot pad, airways, oesophagus, gastrointestinal tract, pancreas, from patients with non-insulin-dependent diabetes mellitus urinary bladder, uterus, and spinal cord (cervical, thoracic, and lumbar (Westermark et al., 1986; Cooper et al., 1987). The peptide is levels). In addition, dorsal root ganglia and spinal cord from newborn rats (day 0; n = 5) were excised. For in situ hybridization, the specimens were frozen on dry ice and stored at -80°C; for immunocyto- Received Apr. 17, 1995; revised July 21, 1995; accepted July 25, 1995. chemistry, the specimens were immersed overnight in 2% paraformal- This study was supported by the Swedish Medical Research Council (Project dehyde and 0.2% picric acid (buffered in 0.1 M phosphate, pH 7.2) 12X-4499 and 5941). The Swedish Diabetes Association, The Heart Lung, followed by repeated rinsing in sucrose-enriched (10%) buffer. The Albert Pahlsson, Wiberg and Crafoord Foundations, The Novo Nordisk Insulin specimens were frozen on dry ice and stored at -80°C until being cut Fund and the Faculties of Medicine, University of Lund and University of Linkoping. We gratefully thank Dr. Amy Percy, Mr. Mark Fineman, and Dr. on a cryostat (10 km). For immunochemistry (radioimmunoassay, RIA) Joy Koda (Amylin Pharmaceuticals Inc., San Diego, CA) for providing F 055. and reverse phase high-performance liquid chromatography (HPLC), 24 and data on antibodv characterization. pooled (n = 10) dorsal root ganglia and specimens from the spinal cord Correspondence should be addressed to Hindrik Mulder, M.D., Department were frozen on dry ice. of Medical Cell Research, University of Lund, Biskopsgatan 5, S-22362 Lund, In situ hybrid&ion. Two 30-mer oligodeoxyribonucleotide probes Sweden. complementary to nucleotides 80-109 and 215-244 in cDNA encoding Copyright 0 1995 Society for Neuroscience 0270.6474/95/157625-08$05.00/O rat IAPP were used (Leffert et al., 1989). These sequences were unique 7626 Mulder et al. - IAPP/Amylin in Sensory Neurons

Table I. Details of antibodies used for immunocytochemistry

Code Antigen Raised in Dilution Source F 055-24 Human IAPP l-10 Monoclonal 1:320 Amylin Pharmaceuticals Inc., San Diego, CA 8425 Rat a-CGRP Rabbit 1:640 Euro-Diagnostica, Malmo, Sweden SP I Substance P Rabbit I:160 Dr. P Emson, MRC Gruop, Cambridge, UK 9223 Nitric oxide synthase Rabbit I:1280 Euro-Diagnostica 88121-3 PACAP l-27 Rabbit 1:640 Dr. A. Arimura, Tulane University School of Medicine, New Orleans, LA N-SOM Somatostatin Rabbit I:800 Inc. Star Corp., Stillwater, MN 8416 Porcine galanin Rabbit 1:320 Euro-Diagnostica

for rat IAPP mRNA (GenBank EMBL, 12/94). Also, a 30.mer oligo- Morphometry. The diameters of nerve cell bodies in dorsal root gan- probe complementary to nucleotides 3839-3868 in rat oi-CGRP cDNA glia labeled by the IAPP probes were determined. Using a calibrated was used (Amara et al., 1984). The probes were 3’.endtailed with “S- scale bar inserted into the eyepiece of the microscope, 100 consecutive dATP as previously described (Mulder et al., 1993). The protocol for nucleated nerve cell bodies from levels C, to L? were measured in in situ hybridization was adopted with modifications from Dagerlind et darkfield. Further, the percentage of IAPP probe-labeled nerve cell bod- al. (1992). Fresh frozen sections (10 km) were thaw mounted onto ies (847 counted) in dorsal root ganglia was determined in two to three chrome-alum-coated slides and stored overnight at -20°C. The slides sections from 12 ganglia from different levels (C, to L,). In addition, were rapidly warmed to room temperature and the hybridization solu- three to four consecutive sections of the two dorsal root ganglia from tion was applied to the sections. The hybridization buffer has previously levels C, to L, were processed for IAPP immunofluorescence. The num- been described (Mulder et al., 1993); the concentration of the IAPP and ber of IAPP-immunoreactive nerve cell bodies in each section was de- CGRP probes were 2.0 and I .O pmol/ml, respectively. The IAPP probes termined, and the frequency at each level calculated. Data on percentage were used separately or in a mix containing an equimolar amount of of IAPP probe-labeled cells and frequency of IAPP-immunoreactive the probes. Prior to hybridization, dithiothreitol was added to the hy- cells are given as mean -t SEM. bridization buffer at a final concentration of 0.2 M. Hybridization was Radioimmunoassay and reverse phase high-petformance liquid chro- carried out overnight in sealed moist chambers at 37°C. After hybrid- matography. The RIA has previously been described in detail (Christ- ization the coverslips were removed by immersion in 1 X saline sodium manson et al., 1993). Briefly, the assay is based on rabbit anti-human citrate (SSC; 0.15 M NaCl, 0.015 M sodium citrate) at room temperature. IAPP antiserum (Peninsula, Merseyside, UK) with labeled rat IAPP The sections were washed in 1 X SSC at 55°C (4X 15 min) and once (Amersham, Solna, Sweden) as tracer; the assay detects changes be- at room temperature (30 min). Finally, the sections were dehydrated and tween adjacent samples equivalent to 80 fmol/gm wet weight tissue. cleared in chloroform, after which they were air dried. For autoradiog- Before assay, dorsal root ganglia and specimens from spinal cord raphy, the slides were dipped in Ilford K-5 emulsion diluted 1: I with [weighing 10 mg (10 pooled ganglia) and 20-50 mg, respectively] were distilled water, air dried, and kept refrigerated at 4°C in light-sealed boiled in 0.5 M acetic acid (10 ml/g wet weight tissue or not less than boxes. After 2 and 4 weeks of exposure (CGRP and IAPP, respectively), 200 ~1); the extracts were assayed in duplicate dilutions equivalent to the autoradiographs were developed in Kodak D- 19 and counterstained 5, 15, and 25 pJtube. Extract samples were also analyzed in a reverse with hematoxylin or methylene blue. For control purposes, sections phase HPLC chromatograph (LKB, Bromma, Sweden). Before chro- were incubated in RNase A (45 (*g/ml; Sigma, St. Louis, MO; 30 min, matography, the samples were centrifuged at 2500 rpm for 5 min. The 37°C) prior to hybridization, As an additional control, a loo-fold molar supernatant (200 ~1) was applied to a TSK gel ODS-120 T (4.6 X 250 excess of unlabeled probe was added to the hybridization buffer. Auto- mm) column (LKB). A 30 min linear gradient of 40-53% solvent B radiographic labeling of nerve cell bodies in the control experiments (see below) was used at a flow rate of 0.7 ml/min. Solvent A was 0.1% was not obtained. trifluoroacetic acid in water and solvent B 30% of solvent A in aceto- Immuno~ytochemistr. The protocols used for indirect immunofluo- nitrile. The effluent was monitored at 226 nm. Fractions (500 ~1) were rescence and double immunofluorescence have previously been de- collected and dried in a Savant Speedvac SC-100 vacuum centrifuge scribed in detail (Mulder et al., 1993); details of the antibodies used are (Techtum Lab, Umeb, Sweden) and reconstituted in 500 ~1 assay buffer given in Table 1. Briefly, the sections were incubated with the primary prior to analysis for IAPP-immunoreactivity in the RIA. Standard rat antibodv overnight at 4°C (48 hr for monoclonal antibodies) followed IAPP (Multiple Peptide Systems, San Diego, CA) and rat a-CGRP were by a secondary intibody, coupled to fluorescein isothiocyanate (FITC) used as controls. and with specificity for immunoglobulin G of the primary antibody. In double immunofluorescence, incubation of the sections with one pri- Results mary antibody was followed by an incubation overnight with a second primary antibody. Thereafter, secondary antibodies, with specificity for In situ hybridization mouse or rabbit immunoglobulin G and coupled to either FITC or tet- The IAPP probes labeled a population of small- to medium-sized ramethyl rhodamine isothiocyanate (TRITC): were applied to the sec- nerve cell bodies in the dorsal root ganglia and the jugular- tions. By shifting the microscope filters, the localization of two primary nodose and trigeminal ganglion (Fig. 1); the CGRP probe la- antibodies in a section was determined. The specificity and possible cross reactivity of the IAPP and CGRP antibodies were checked by beled a greater number of nerve cell bodies, which were of small preabsorption with human IAPP and rat a-CGRP prior to incubation. to large size (Fig. 1B). Both IAPP probes labeled the nerve cell The antigen was added to the working dilution of the antibody at a bodies in the ganglia to the same extent, but the density of la- concentration of IO-100 &ml. Preabsorption of the IAPP antibody beling increased when the probes were used as a mix. In the with human IAPP quenched all immunostaining, whereas it was not affected by preabsorption with rat o-CGRI? Preabsorption of the CGRP dorsal root ganglia, 24 ? 1.4% of all nerve cell bodies were antibody with rat or-CGRP quenched all immunostaining, whereas it was labeled by the IAPP probes. The mean diameter of nerve cell not affected by preabsorption with rat IAPP bodies was 30 + 0.7 km; the distribution of cell diameters is The Journal of Neuroscience, November 1995, 75(11) 7627

35 T

20 25 30 35 40 45 50 55

DlAMETEFl(pm) Figure 2. Diametersof nervecell bodiesin dorsalroot ganglialabeled by the IAPP probes.The percentageof differentdiameters are given. IAPP mRNA is predominantlyfound in small-to medium-sizednerve cell bodieswith a meandiameter of 30 + 0.7 urn.

shown in Figure 2. Hybridization with the IAPP and CGRP probes in adjacent sections from dorsal root ganglia revealed that the IAPP probeslabeled a subpopulationof nerve cell bodies labeledby the CGRP probe. In the spinal cord, motoneurons were labeled by the CGRP probe but not by the IAPP probes.

Immunocytochemistry In the adult rat, a moderate number of IAPP-immunoreactive nerve cell bodies was seenin the dorsal root ganglia and the jugular-nodose and trigeminal ganglion (Fig. 3A,C,E). The IAPP-containing nerve cell bodies, which were predominantly of small to medium size, were scatteredand more numerousin the dorsalroot ganglia than in the jugular-nodoseand trigeminal ganglion; the frequency of IAPP-immunoreactive neurons at each level from C, to L, is shown in Figure 4, demonstrating that the IAPP-containing nerve cell bodies were most frequent at the lower thoracic levels. In the dorsal root ganglia, double immunofluorescencerevealed that IAPP occurred in a subpopulation of CGRP-containing nerve cell bodies (Fig. 3A,B). Fur- ther, IAPP-like immunoreactivity was seenin a majority of the nerve cell bodies containing substanceP (SP; Fig. 3C,D). In addition, a few nerve cell bodies containing IAPP also stored pituitary adenylate cyclase-activating polypeptide (PACAP). IAPP was not observed in nerve cell bodiescontaining somatostatin, galanin, or nitric oxide synthase.In the dorsal root gan- Figure 1. In situ hybridization in sensory ganglia. The IAPP probes demonstrate the presence of IAPP mRNA in a population of small- to glia, nerve fibers were seen containing IAPPKGRP and medium-sized nerve cell bodies (arrows) in dorsal root ganglion (A) IAPP/SP (Fig. 3A-D). In dorsalroot ganglia from newborn rats and the trigeminal ganglion (C). CGRP mRNA is present in a greater (day 0), IAPP-immunoreactive nerve cell bodies were present; number of nerve cell bodies in dorsal root ganglion, including those of they were identical to the onescontaining CGRP Also, in the large size (B; arrowheads). Bar, 20 km. jugular-nodoseganglion, a small population of IAPP-immunoreactive nerve cell bodies and fibers were seen, constituting a subpopulationof those storing CGRP (Fig. 3E,F). A dense plexus of IAPP-immunoreactive nerve fibers was found in the superficial layers of the dorsal horns in the spinal cord from both adult and newborn rats (Fig. 5A). Thesefibers 7626 Mulder et al. * IAPP/Amylin in Sensory Neurons

Figure 3. Double immunofluorescence. Dorsal root ganglion (A-D); jugular-nodose ganglion (E and F). A moderate number of IAPP-immunoreactive nerve cell bodies is seen (A and C). IAPP occurs in a subpopulation of CGRP-containing neurons (A and B; arrows), a number of CGRP- containing neurons lacking IAPP (B; arrowheads). Also, note the coexistence of IAPPEGRP in nerve fibers within the ganglia (A and B; small arrows). IAPP occurs in a majority of the nerve cell bodies that store SP (C and D; arrows), although some IAPP-containing nerve cell bodies lack SP (C; arrowheads). In addition, IAPP coexists with SP in a population of nerve fibers (C and D; small arrows). In the jugular-nodose ganglion, a few IAPP-immunoreactive nerve cell bodies and fibers are seen (E; large and small urrows, respectively), constituting a subpopulation of those containing CGRP (F); many CGRP-immunoreactive nerve cell bodies and nerve fibers lack IAPP (F; large and small arrows, respectively). Bar, 20 pm.

contained in addition CGRP (Fig. 5B) and/or SP; the fibers con- fibers. In contrast, immunoreactive CGRP was present in nerve taining CGRP were more numerousthan the ones containing fibers throughout the gastrointestinaltract (Fig. 5F) and in nerve IAPP whereas those containing SP where approximately equal cell bodiesin the enteric ganglia. However, endocrinecells con- in number to those containing IAPP The posterolateraltract of taining IAPP were encounteredin the gastrointestinaltract, be- Lissauerdisplayed dense CGRP-immunoreactivity, whereasonly ing most abundant in the antrum-pylorus (Fig. 5E); endocrine a sparsesupply of IAPP-immunoreactive nerve fibers was seen cells containing CGRP were not observed (Fig. 5F). In pancre- (Fig. 5AJ3). Motoneurons lacked IAPP-immunoreactivity while atic islets, IAPP-immunoreactivity was confined to islet cells, containing CGRP which predominatedcentrally in the islets. The CGRP antibody In the basal parts of the epithelium covering the nose and revealed peripherally located islet cells, containing, in addition, tongue (Fig. 5C), a few, delicate IAPP-containing fibers were somatostatinand IAPP, as well as scatterednerve fibers in the seen.Except for a few IAPP-immunoreactive fibers in the py- islets, whereasIAPP-immunoreactive nerve fibers were lacking. loric sphincter (Fig. 5E) and in the duodenalglands of Brunner, In the urinary bladder,a small subepithelialpopulation of IAPP- the gastrointestinaltract was devoid of IAPP-containing nerve containing fibers was seen.In the uterus, a few IAPP-immuno- The Journal of Neuroscience, November 1995, 15(11) 7629

thermore, the concentration of IAPP in extracts from dorsal root ganglia and spinal cord was low (3-5 pmol/g), as compared to that previously reported for CGRP (approximately 500 pmol/g; Mulderry et al., 1988). In the ganglia and tissues examined, in which IAPP occurred in neuronal elements, expression of IAPP was seen in a subpopulation of CGRP-containing neurons; IAPP mRNA was present in 24% of the neurons in dorsal root ganglia, whereas CGRP mRNA occurs in 3746% of the neurons in dorsal root ganglia (Noguchi et al., 1993; Mulder et al., 1994b). In certain tissues heavily innervated by CGRP, such as the gastrointestinal tract and airways, IAPP occurred sparsely or was lacking. However, as previously demonstrated (Mulder et al., 1994a), IAPP occurred in gastrointestinal endocrine cells, whereas CGRP was absent from such cells. The colocalization of IAPP and CGRP was not entirely surprising, since the two peptides have probably evolved due to a duplication of an ancestral gene (Nishi et al., 1989). It is not inconceivable that the two genes through evo- lution have retained some common regulatory elements that de- termine the site of their expression; in this case, a population of sensory neurons. In neuronal tissues, it is not uncommon that Figure 4. Frequency of IAPP-immunoreactive nerve cell bodies at structurally related peptides with similar biological effects are different levels of dorsal root ganglia. The IAPP-immunoreactive cell bodies are present at all levels investigated (data from L, lacking), being colocalized. Thus, SP and neurokinin A are coexpressed in sen- most abundant at the lower thoracic levels. sory neurons, the peptides deriving from the same gene and having similar biological effects (Sundler et al., 1985). In addition, IAPP was colocalized with SP and PACAP, which are reactive fibers occurred among the smooth muscle bundles. All both known to coexist with CGRP in sensory neurons (Sundler _ IAPP fibers seen in these tissues contained, in addition, CGRP et al., 1985; Moller et al., 1993). (Fig. 5QF); the innervation by CGRP was exceedingly more A major concern in our study was to establish the molecular prominent with a marked perivascular occurrence, where IAPP, and chemical identity of IAPP mRNA and IAPP in sensory neu- in contrast, was absent. IAPP was not encountered in the air- rons, since the structurally related neuropeptide CGRP (Wester- ways, which displayed an abundant CGRP innervation. mark et al., 1986; Cooper et al., 1987) is ubiquitously expressed in the sensory nervous system (Sundler et al., 1985; Ishida-Ya- Immunochemistry and reverse phase high-performance liquid mamoto and Tohyama, 1989). The IAPP probes used for in situ chromatography hybridization lacked sequence homology with CGRP mRNA. In extracts from pooled dorsal root ganglia and different levels Further, the labeling of the two IAPP probes was identical in the of the spinal cord, the concentration of IAPP was 3.2 pmol/g ganglia, and the control experiments were negative. Collectively, and 4.7 -C 2.1 pmol/g wet weight tissue, respectively. Reverse this ensured a high degree of specificity in the hybridization phase HPLC on extracts from dorsal root ganglia and spinal cord experiments. The immunostaining of nerve cell bodies or fibers revealed that the immunoreactivity eluted in two peaks; the first was not affected by preabsorption of the monoclonal IAPP an- peak was smaller and eluted simultaneously with authentic rat tibody with CGRP, making a crossreaction with CGRP unlikely. IAPP, whereas the second corresponded to authentic rat ol-CGRP Moreover, IAPP-like immunoreactivity was absent from several (Fig. 6). The elution profiles were identical in the extracts from sites displaying CGRP-like immunoreactivity and, even in the dorsal root ganglia and spinal cord, and no other immunoreac- same section, CGRP-containing neurons were seen to lack IAPP tive peaks appeared. These observations support the presumption that the monoclonal IAPP antibody does not recognize CGRP Reverse phase HPLC Discussion on extracts from dorsal root ganglia and spinal cord yielded one In this study, we present evidence that IAPP is expressed in small peak coeluting with authentic rat IAPP and one larger peak sensory neurons. Thus, besides being an islet hormone candi- coeluting with rat ol-CGRP This is explained by the fact that the date, IAPP is also a putative sensory neuropeptide. IAPP was polyclonal antibody used in thk RIA crossreacts slightly with expressed in a population of small- to medium-sized neurons in CGRP (approximately 0.4%; unpublished result), which is pres- the trigeminal, jugular-nodose, and dorsal root ganglia. The sen- ent in these tissues at a much higher concentration than IAPP sory nature of IAPP was confirmed by the finding of IAPP- (see above). Although the exact concentration of IAPP in the immunoreactive neurons forming a dense plexus in the superfi- dorsal root ganglia and spinal cord remains unresolved, con- cial layers of the dorsal horns in the spinal cord, in a manner ceivably being low (in the range of l-2 pmol/g), the chromato- similar to that of CGRP and other sensory neuropeptides (Sun- graphic data are in agreement with the hybridization and im- dler et al., 1985). Also, in peripheral tissues with a prominent munocytochemical findings and, thus, further support that IAPP sensory innervation, a sparse supply of IAPP-containing fibers is expressed in the dorsal root ganglia and spinal cord. was encountered. The level of IAPP mRNA in nerve cell bodies At this point, one can only speculate on the role of IAPP in conceivably was low, since the exposure time for the IAPP sensory neurons. IAPP was expressed in part in sensory neurons probes and probe concentrations were twice that of the CGRP of small size; such neurons give rise to C-type unmyelinated probe, while still yielding a weaker labeling of neurons. Fur- fibers, which are known to convey nociceptive transmission to 7630 Mulder et al. l IAPP/Amylin in Sensory Neurons

Figure 5. Double immunofluorescence. In the spinal cord (A and B), IAPP-immunoreactive neurons form a plexus in the superficial layers of the dorsal horns (A). These fibers have a similar distribution to those containing CGRP (B), but are fewer and less intensely stained. Note the scarcity of immunoreactive IAPP in Lissauer’s tract (A; arrow), which displays a dense innervation by CGRP (B; arrow). In the tongue (C and D), a sparse supply of IAPP-immunoreactive nerve fibers runs beneath the epithelium and encircles a papilla (C, arrows); these fibers constitute a subpopulation of CGRP-containing nerve fibers (D; arrows). The pyloric musculature displays a few delicate IAPP-containing nerve fibers (E; small arrows), constituting a subpopulation of CGRP-containing nerve fibers (F; small arrows). Note that some CGRP-fibers (F, arrowheads) lack IAPI? IAPP- immunoreactive endocrine cells are present in the mucosa (E, large arrow); these cells are devoid of CGRP-immunoreactivity. Bars: A and B, 100 pm; C and D, 50 pm; E and F, 50 pm. The Journal of Neuroscience, November 1995, 75(11) 7631

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